Division of Allergy and Immunology and Joy McCann Culverhouse Airway Disease Center, Department of Internal Medicine, University of South Florida College of Medicine and James A, Haley Veteran's Hospital, Tampa, FL 33612, USA.

ABSTRACT Chitosan, a polymer derived from chitin, has been used for nasal drug delivery because of its biocompatibility, biodegradability and bioadhesiveness. Theophylline is a drug that reduces the inflammatory effects of allergic asthma but is difficult to administer at an appropriate dosage without causing adverse side effects. It was hypothesized that adsorption of theophylline to chitosan nanoparticles modified by the addition of thiol groups would improve theophylline absorption by the bronchial epithelium and enhance its anti-inflammatory effects.
We sought to develop an improved drug-delivery matrix for theophylline based on thiolated chitosan, and to investigate whether thiolated chitosan nanoparticles (TCNs) can enhance theophylline's capacity to alleviate allergic asthma.
A mouse model of allergic asthma was used to test the effects of theophylline in vivo. BALB/c mice were sensitized to ovalbumin (OVA) and OVA-challenged to produce an inflammatory allergic condition. They were then treated intranasally with theophylline alone, chitosan nanoparticles alone or theophylline adsorbed to TCNs. The effects of theophylline on cellular infiltration in bronchoalveolar lavage (BAL) fluid, histopathology of lung sections, and apoptosis of lung cells were investigated to determine the effectiveness of TCNs as a drug-delivery vehicle for theophylline.
Theophylline alone exerts a moderate anti-inflammatory effect, as evidenced by the decrease in eosinophils in BAL fluid, the reduction of bronchial damage, inhibition of mucus hypersecretion and increased apoptosis of lung cells. The effects of theophylline were significantly enhanced when the drug was delivered by TCNs.
Intranasal delivery of theophylline complexed with TCNs augmented the anti-inflammatory effects of the drug compared to theophylline administered alone in a mouse model of allergic asthma. The beneficial effects of theophylline in treating asthma may be enhanced through the use of this novel drug delivery system.

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Abstract The present study reports the preparation and physicochemical characterization of surface-modified poly(lactide-co-glycolide) (PLGA) microparticles containing interleukin-2 (rhIL-2) for pulmonary delivery. The surface of the microparticles was modified with mucoadhesive polymers such as chitosan and Carbopol 971P. The feasibility of this surface modification was confirmed by measuring the zeta potential. Chitosan-modified PLGA microparticles showed a positive zeta potential, while Carbopol-modified PLGA microparticles were negatively charged. The mucin binding efficiency values have shown that the positively charged chitosan coated microparticles showed a higher adhesive percent to the mucin than the negatively charged un-coated or Carbopol 971P coated microparticles. Furthermore, surface modification of microparticles with chitosan and Carbopol 971P has yielded a slight decrease in the amount of protein initially released. These findings suggest the suitability of surface-modified PLGA microparticles as an efficient carrier system for delivery peptides and proteins to the respiratory tract.

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Topical formulations, commonly applied for treatment of anterior eye diseases, require frequent administration due to rapid clearance from the ocular surface, typically through the lacrimal drainage system or through over-spillage onto the lids. We report on a mucoadhesive nanoparticle drug delivery system that may be used to prolong the precorneal residence time of encapsulated drugs. The nanoparticles were formed from self-assembly of block copolymers composed of poly(d, l-lactide) and Dextran. The enhanced mucoadhesion properties were achieved by surface functionalizing the nanoparticles with phenylboronic acid. The nanoparticles encapsulated up to 12 wt.% of Cyclosporine A (CycA) and sustained the release for up to five days at a clinically relevant dose, which led us to explore the therapeutic efficacy of the formulation with reduced administration frequency. By administering CycA-loaded nanoparticles to dry eye-induced mice once a week, inflammatory infiltrates were eliminated and the ocular surface completely recovered. The same once a week dosage of the nanoparticles also showed no signs of physical irritation or inflammatory responses in acute (1 week) and chronic (12 weeks) studies in healthy rabbit eyes. These findings indicate that the nanoparticles may significantly reduce the frequency of administration for effective treatment of anterior eye diseases without causing ocular irritation.

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The use of nanoparticles (NPs) can improve drug delivery in many pharmaceutical applications. Compared to conventional compounds, NPs are expected to show better tissue penetration and to deliver higher drug amounts more selectively to the target site. NP-based formulations are most advanced for parenteral applications but they are also used for delivery across mucus-covered epithelial surfaces (eye, orogastrointestinal tract, airways, and vagina). The mucus layer represents an important barrier for an NP-based delivery system. In this review, mucus composition, architecture, and turnover of the mucus layer at different anatomical locations are addressed. The influence of particle parameters on mucus penetration/permeation is mentioned and examples for mucoadhesive, mucolytic, and mucus-penetrating particle systems are listed. While mucoadhesive particles have a relatively long history in NP-based drug delivery, mucus penetrating NPs have been developed more recently. These particles may be advantageous for drug delivery to anatomical sites with high mucus turnover.

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Respiratory Research 2006, 7:112 http://respiratory-research.com/content/7/1/112Page 2 of 10(page number not for citation purposes)BackgroundAsthma is a chronic inflammatory disease of the airwaycharacterized by the infiltration of eosinophils, epithelialhyperplasia leading to hypersecretion of mucus and thepresence of airway hyperresponsiveness (AHR) to a vari-ety of stimuli [1,2]. Theophylline had been used world-wide for the treatment of asthma for several decades, butits use has recently declined owing to the increased use ofinhaled glucocorticoids. Theophylline's side effects, suchas nausea, headache and cardiac arrhythmias, at the dosenecessary to achieve bronchodilation (plasma levels of 10to 20 mg/L) in asthma also limit its use [1,3]. In spite ofthese drawbacks, theophylline remains a widely pre-scribed anti-asthmatic agent [4-6].In addition, theophylline has been reported to suppressthe activation of inflammatory cells, such as neutrophilsand eosinophils at concentrations lower than what isrequired for bronchodilation [3,7,8]. Recent clinical andexperimental research showed that theophylline atplasma levels of <10 mg/L still possesses anti-inflamma-tory and immunomodulatory properties, which may per-mit its use in the long-term treatment of chronicobstructive pulmonary inflammation [9,10]. The inci-dence of adverse side effects is minimized at this dose [1].Chitosan, a linear polysaccharide derived from chitinobtained from crustacean shells, has emerged as a usefuldrug delivery matrix because of it polycationic nature, bio-degradability, biocompatibility, mucoadhesiveness andease of physical and chemical modification [11]. Theinteraction between cationic amino groups on chitosanand anionic moieties such as sialic and sulfonic acids onthe mucus layer is responsible for its mucoadhesiveness[12]. In addition, chitosan enhances epithelial permeabil-ity through the opening of tight junctions between epithe-lial cells [13]. Recently, it was reported that the covalentattachment of thiol groups to polymers greatly increasestheir mucoadhesiveness and permeation properties with-out affecting biodegradability [12,14].In this study, we hypothesized that the absorption of the-ophylline through bronchial mucosa could be enhancedby administration with thiolated chitosan nanoparticles(TCNs) because of their greater mucoadhesiveness andpermeability properties. The anti-inflammatory effects oftheophylline were measured in BALB/c mice that hadbeen made allergic to ovalbumin. Histopathology of lungtissue after OVA challenge, eosinophilia in bronchoalveo-lar lavage fluid, levels of mucin production and apoptosisof lung cells were examined to evaluate the effects of the-ophylline.Materials and methodsMaterialsChitosan (33 kDa) was obtained from TaeHoon Bio. Co(Korea) and used as received. The viscosity and degree ofdeacetylation as determined by the supplier were 2.8 cps(0.5% solution in 0.5% acetic acid at 20°C) and 90%,respectively. Thioglycolic acid, sodium tripolyphosphate,theophylline and mucin type I-S (9–17% sialic acid) andIII (~1% sialic acid) were purchased from Sigma-Aldrich,USA, and used without further purification.Synthesis of thiolated chitosanThe chemical modification of chitosan was performed aspreviously described [15,16]. Chitosan (500 mg) was dis-solved in 50 mL of 1.0% acetic acid. In order to facilitatereaction with thioglycolic acid (TGA), 100 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride(EDAC) was added to the chitosan solution. After EDACwas dissolved, 30 mL of TGA was added and the pH wasadjusted to 5.0 with 3 N NaOH. The reaction mixture wasstirred and left for 3 h at room temperature. To eliminatethe unbound TGA and to isolate the polymer conjugates,the reaction mixture was dialyzed against 5 mM HCl fivetimes (molecular weight cut-off 10 kDa) over a period of3 days in the dark, then two times against 5 mM HCl con-taining 1.0% NaCl to reduce ionic interactions betweenthe cationic polymer and the anionic sulfhydryl com-pound.Preparation and characterization of chitosan nanoparticlesChitosan suspensions of 0.2% (w/v) were prepared in 1%acetic acid. Sodium tripolyphosphate (TPP, 1.0%) wasadded dropwise to 6 ml of chitosan with stirring, followedby sonication with a Dismembrator (Fisher Scientific) for10 sec at a power setting of 3 watts. The resulting chitosanparticle suspension was centrifuged at 10,000 × g for 10min. The pelleted particles were resuspended in deionizedwater with 10 sec sonication and lyophilized. The meansize and zeta potential of the chitosan nanoparticles weredetermined by photon correlation spectroscopy using aZetaPlus particle analyzer (Brookhaven Instrument Corp.,Holtsville, NY, USA).Adsorption of mucin by chitosan nanoparticles and mucin assayMucoadhesiveness was calculated as the amount of mucinadsorbed by 2 mg of chitosan nanoparticles in a certaintime period. Chitosan nanoparticle suspensions (4 mg/mL) were mixed with type I-S or type III mucin solutions(0.5 and 1 mg/mL), vortexed, and incubated at 37°C for1, 6, 12 and 18 h. After adsorption, the suspensions werecentrifuged at 10,000 × g for 10 min and free mucin wasmeasured in the supernatant by a colorimetric methodusing periodic acid/Schiff (PAS) staining [17]. Schiff rea-

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Respiratory Research 2006, 7:112 http://respiratory-research.com/content/7/1/112Page 3 of 10(page number not for citation purposes)gent was prepared by diluting pararosaniline solution (40g/L in 2 M HCl, Sigma) with water to give a final concen-tration of 1.0%. Sodium bisulfite (80 mg) was added to 5mL of Schiff reagent and the resultant solution was incu-bated at 37°C until it became colorless or pale yellow.Periodic acid solution was freshly prepared by adding 10µL of 50% periodic acid to 7 mL of 7% acetic acid. Super-natants were mixed with 100 µL of dilute periodic acidand incubated for 2 h at 37°C. Then, 100 µL of Schiff rea-gent was added at room temperature, and after 30 min theabsorbance was measured at 560 nm. The amount ofmucin adsorbed by the chitosan nanoparticles was deter-mined by subtracting the concentration of mucin in solu-tion after adsorption from that before. Mucin standards(0.1, 0.25 and 0.5 mg/mL) were measured by the sameprocedure and a standard calibration curve was prepared.Nasal delivery of theophyllineBALB/c mice (n = 4) were sensitized by i.p. injection of 50µg ovalbumin (OVA, Sigma Grade V,) precipitated withalum (Imject, Pierce) on day 1. On day 8 and 14 micewere challenged intranasally with 50 µg of OVA. Theo-phylline (30 mM) was added to the chitosan nanoparticlesuspension (4 mg/mL) and allowed to adsorb to the nan-oparticles for 12 h at room temperature. Mice were given50 µL of theophylline with or without chitosan nanopar-ticles intranasally on days 15, 16 and 17. Control micewere given PBS only.Determination of eosinophil number in bronchoalveolar lavage (BAL) fluidMice were euthanized on day 22 and lungs were lavagedwith 500 µL of PBS introduced through the trachea. BALfluid was centrifuged at 300 × g for 5 min, rinsed and cellsresuspended with PBS. Aliquots of the cell suspensionwere applied to slides by Cytospin (Shandon Scientific) at500 × g for 5 min allowed to air dry and stained with mod-ified Wright's stain (Hema-3, Fisher Scientific). The eosi-nophils were determined morphologically and aminimum of 200 cells per slide were counted under themicroscope.Lung histology, mucin production and apoptosis assayMice were euthanized and their lungs were perfused withPBS, removed and fixed in 4% buffered formalin. Lungswere embedded in paraffin, sectioned and stained withhematoxylin and eosin. A semi-quantitative microscopicevaluation of inflammatory cells in the entire lung sectionwas done by persons blinded to the type of treatment.Inflammatory infiltrates were assessed morphologicallyfor location, numbers and cell types [18,19]. Epithelialchanges were scored as: 0 = no change, 1 = increased epi-thelial cell cytoplasm without desquamation, 2 = epithe-lial desquamation without bronchial exudates composedof inflammatory cells, 3 = bronchial exudates composedof desquamated epithelial cells and inflammatory cells.For peribronchovascular infiltrates: 0 = no infiltrate, 1 =infiltrate up to 4 cells thick in most vessels, 2 = infiltratefrom five to seven cells thick in most vessels, 3 = infiltrategreater than 7 cells thick in most vessels. For interstitial-alveolar cell infiltrates: 0 = no infiltrate, 1 = mild general-ized increase in cell mass of the alveolar septa withoutthickening of the septa or significant airspace consolida-tion, 2 = dense septal mononuclear infiltrates with thick-ening of septa, 3 = significant alveolar consolidation inaddition to interstitial inflammation.Goblet cells were identified using a monoclonal antibodyto the marker protein mucin 5AC (MUC5AC, Lab VisionCo) visualized immunohistochemically. The presence ofapoptotic cells was determined by examining sectionsusing the TUNEL (terminal deoxynucleotidyl transferasedUTP nick end-labeling) assay. Lung sections from paraf-fin blocks were dewaxed in xylene, rehydrated to PBS andfixed with 4% paraformaldehyde for 15 min at room tem-perature. Sections were washed three times in PBS, perme-ablized for 15 min with 0.1% Triton X-100 and incubatedwith the TUNEL reagent at 37°C for 1 h. The reaction wasterminated by rinsing slides once with 2× SSC and threetimes in PBS. The lung sections were examined under afluorescent microscope and photographed.Statistical analysisPairs of groups were compared by Student's t test. Differ-ences between groups were considered significant at p <0.05. Values for all measurements are expressed as means± SD.ResultsCharacterization of thiolated chitosan nanoparticlesLyophilized thiolated chitosan (chitosan-thioglycolic acidconjugates, Fig. 1) is a fibrous white powder easily solublein water. The degree of thiolation was determined byreacting the thiol groups with Ellman's reagent (5,5'-dithiobis(2-nitrobenzoic acid) and reading the absorb-ance at 412 nm spectrophotometrically. The content ofthiol groups on thiolated chitosan was found to be 17–30µM/g. Unmodified and thiolated chitosan nanoparticlesprepared by ionic crosslinking with sodium tripolyphos-phate have a diameter of 220 ± 23 nm. The zeta potentialis a measure of the charge density on particles and canaffect their capacity for aggregation and interaction withcharged surfaces such as cells or membranes. Because ofits amino groups, unmodified chitosan has a net positivecharge and zeta potential of 22.7 ± 4 mV. Thiolationreduces the charge somewhat so that TCNs have a zetapotential of 15.3 ± 2 mV.

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Respiratory Research 2006, 7:112 http://respiratory-research.com/content/7/1/112Page 4 of 10(page number not for citation purposes)Mucoadhesiveness of chitosan nanoparticlesMucin adsorption by TCNs was measured as a way to testtheir ability to bind to the mucosal surfaces of the body(mucoadhesiveness). Mucin, a glycoprotein, is the majorcomponent of the mucus that coats the cells lining the sur-faces of the respiratory, digestive and urogenital tracts[20]. Mucoadhesiveness was calculated as the amount ofmucin adsorbed by 2 mg of chitosan nanoparticles in aChemical structure of chitosan-thioglycolic acid conjugate (A) and transmission electron micrograph of chitosan nanoparticles following ionic crosslinking (B)Figure 1Chemical structure of chitosan-thioglycolic acid conjugate (A) and transmission electron micrograph of chitosan nanoparticles following ionic crosslinking (B). Adsorption kinetics of mucin I-S with unmodified and thiolated chitosan nanoparticles (C). The value of mucin adsorbed represents the amount of mucin per 2 mg of chitosan nanoparticles. The experiments were repeated twice and results are expressed as mean ± S.D (**P < 0.01, *P < 0.05 relative to 1 h).

Respiratory Research 2006, 7:112 http://respiratory-research.com/content/7/1/112Page 5 of 10(page number not for citation purposes)certain time period. Unmodified chitosan nanoparticlesadsorbed a larger amount of mucin I-S and mucin III,compared to thiolated chitosan nanoparticles after 1 hincubation (data not shown). The amount of mucinadsorbed increased with mucin concentration and per-centage of sialic acid residues. Both unmodified and thi-olated chitosan nanoparticles bound more mucin type I-S(containing 12% sialic acid) than mucin III (containing1% sialic acid). Unmodified chitosan nanoparticlesreached adsorption equilibrium at about 1 h at 37°C. Incontrast, TCNs exhibited a more gradual increase inmucin I-S adsorption and greater binding at 12 h thanunmodified chitosan (Fig. 1c).In vivo effectiveness of thiolated chitosan delivery of theophylline: treatment reduces the number of eosinophils in bronchoalveolar lavage (BAL) fluidThe hypothesis of this study was that the absorption oftheophylline by the bronchial epithelium and its pharma-ceutical action can be enhanced by delivering theophyl-line as a complex with TCNs. To test this, mice allergic toovalbumin (OVA) were challenged with OVA and thengiven theophylline or theophylline complexed with chi-tosan nanoparticles according to the protocol in Fig. 2a.Eosinophils are known to migrate to the site of an allergicreaction and to modulate the allergic inflammatoryresponse [21]. Fig. 2b shows the effect of theophyllinedelivery on the percentage of eosinophils in BAL fluid.Administration of OVA increased the number of eosi-nophils in the airway at the subepithelial region beneaththe basement membrane compared to unchallenged con-trols. The percentage of eosinophils in the BAL fluid wassignificantly reduced in the group treated with theophyl-line plus TCNs compared to untreated mice, or mice givenunmodified chitosan, theophylline or theophylline plusunmodified chitosan.Thiolated chitosan nanoparticles (TCNs) enhance anti-inflammatory effects of theophyllineThe effects of theophylline on OVA allergen-induced his-topathology in lung sections from treated mice are shownin Fig. 3. The lung sections from OVA-challenged allergicmice clearly show epithelial damage, luminal narrowingdue to airway wall edema and obstruction with excessmucus which are typical of bronchial inflammation (Fig.3b). Administration of chitosan or thiolated chitosannanoparticles to the sensitized and challenged mice didnot produce anti-inflammatory effects, however, theo-phylline treatment produced a moderate reduction inlung pathology. The mice treated with theophylline plusTCNs, however, showed a considerable reduction in pul-monary inflammation, decreased epithelial damage,reduced goblet cell hyperplasia and fewer infiltratinginflammatory cells in the interstitial and peribronchovas-cular regions compared to the other groups (Fig. 3g).Unstained lung sections were examined for expression ofmucin by staining with antibody against MUC5AC (Fig.Experimental protocol used for ovalbumin-induced allergic asthma (A) and reduction of eosinophils in BAL fluid from theo- phylline (TPL)-treated mice (B)Figure 2Experimental protocol used for ovalbumin-induced allergic asthma (A) and reduction of eosinophils in BAL fluid from theo-phylline (TPL)-treated mice (B). BAL was performed and eosinophils were counted on day 22. The results of a representative experiment of two performed is shown. Cell counts were performed by different persons in a blinded fashion. CS denotes chi-tosan. (**p < 0.01, *p < 0.07 relative to TPL-thioCS).01020304050ControlUntreatedPure CSThio CSTPL TPL+pureCSTPL+ThioCSPercentage eosinophils*********BATheophylline (i.n)OVA (i.p)81TreatmentDay22OVA (i.n)Sacrifice (BAL, lungs)21OVA (i.n)OVA (i.n)14151617